Showing papers on "Protoplast published in 2017"

Highly efficient mesophyll protoplast isolation and PEG-mediated transient gene expression for rapid and large-scale gene characterization in cassava (Manihot esculenta Crantz).

Abstract: Cassava (Manihot esculenta Crantz) is a major crop extensively cultivated in the tropics as both an important source of calories and a promising source for biofuel production. Although stable gene expression have been used for transgenic breeding and gene function study, a quick, easy and large-scale transformation platform has been in urgent need for gene functional characterization, especially after the cassava full genome was sequenced. Fully expanded leaves from in vitro plantlets of Manihot esculenta were used to optimize the concentrations of cellulase R-10 and macerozyme R-10 for obtaining protoplasts with the highest yield and viability. Then, the optimum conditions (PEG4000 concentration and transfection time) were determined for cassava protoplast transient gene expression. In addition, the reliability of the established protocol was confirmed for subcellular protein localization. In this work we optimized the main influencing factors and developed an efficient mesophyll protoplast isolation and PEG-mediated transient gene expression in cassava. The suitable enzyme digestion system was established with the combination of 1.6% cellulase R-10 and 0.8% macerozyme R-10 for 16 h of digestion in the dark at 25 °C, resulting in the high yield (4.4 × 107 protoplasts/g FW) and vitality (92.6%) of mesophyll protoplasts. The maximum transfection efficiency (70.8%) was obtained with the incubation of the protoplasts/vector DNA mixture with 25% PEG4000 for 10 min. We validated the applicability of the system for studying the subcellular localization of MeSTP7 (an H+/monosaccharide cotransporter) with our transient expression protocol and a heterologous Arabidopsis transient gene expression system. We optimized the main influencing factors and developed an efficient mesophyll protoplast isolation and transient gene expression in cassava, which will facilitate large-scale characterization of genes and pathways in cassava.

Yeast extract elicitation increases vinblastine and vincristine yield in protoplast derived tissues and plantlets in Catharanthus roseus

Abstract: Catharanthus roseus (L.) G. Don, Apocynaceae, is an immensely important medicinal plant, produces a variety of anticancerous compounds. The yield of two most investigated alkaloids vinblastine and vincristine is unfortunately very low. A vast array of technologies including elicitation have recently been used to enrich Catharanthus alkaloid in culture. Yeast extract is a biotic elicitor, the polysaccharide and the peptide moiety have been recognized as a signalling element in enriching secondary metabolites. In this study, the yeast extract elicitation on vinblastine and vincristine was studied in various protoplast derived tissues and plantlets. Four different yeast extract treatments (T1 = 0.5 g/l, T2 = 1.0 g/l, T3 = 1.5 g/l and T4 = 2.0 g/l) were prepared and used. The alkaloid was quantified and a comparative account of yield were presented by the use of High performance thin layer chromatography. The yeast extract amendment in medium improved vinblastine and vincristine yield in cultivating tissues, maximum being in germinating embryos and in in vitro raised leaf. The highest yield was in T3 (1.5 mg/l) in which 22.74% vinblastine and 48.49% vincristine enrichment was noted in germinating embryos; the enhancement was however, treatment-specific. Antioxidant enzymes such as superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase activities were investigated as addition of yeast extract caused cellular stress and had enriched level of alkaloids.

Efficient isolation of Magnolia protoplasts and the application to subcellular localization of MdeHSF1.

Abstract: Magnolia is a woody ornamental plant, which is widely used in urban landscaping However, its lengthy juvenile period and recalcitrance to regeneration impedes functional characterization of its genes We developed an efficient protoplast isolation and transient expression system for Magnolia denudata × Magnolia acuminata ‘Yellow River’ The highest yield of protoplasts was obtained from young leaves digested in 3% Cellulase R10, 08% Macerozyme R10, 004% pectinase and 04 M mannitol enzymolysis solution for 6 h For transfection of protoplasts, 20% PEG4000 for 5 min was optimal To verify the protoplast system and begin to understand heat tolerance in Magnolia, a heat shock transcription factor MdeHSF1 was cloned from ‘Yellow River’, which belongs to the HSF subfamily A and has significant homology with AtHSFA1A Subcellular localization analysis indicated that MdeHSF1 was expressed in the cell nucleus Furthermore, qPCR analysis of the MdeHSF1 transcript level in response to high temperature stress suggested that MdeHSF1 might be involved in regulating heat stress tolerance in ‘Yellow River’ The described protocol provides a simple and straightforward method for isolating protoplast and exploring gene subcellular localization of MdeHSF1 in Magnolia This expands the new research of protoplast isolation and transfection in Magnolia

An efficient protocol for perennial ryegrass mesophyll protoplast isolation and transformation, and its application on interaction study between LpNOL and LpNYC1

Abstract: Perennial ryegrass (Lolium perenne L.) is an important temperate grass used for turf and forage purposes. With the increasing accumulation of genomic and transcriptomic data of perennial ryegrass, an efficient protoplast and transient gene expression protocol is highly desirable for in vivo gene functional studies in its homologous system. In this report, a highly efficient protoplast isolation (5.6 × 107 protoplasts per gram of leaf material) and transient expression (plasmid transformation efficiency at 55.2%) was developed and the detailed protocol presented. Using this protocol, the subcellular locations of two ryegrass proteins were visualized in chloroplasts and nuclei, respectively, and protein–protein interaction between two chlorophyll catabolic enzymes (LpNOL and LpNYC1) was recorded in its homologous system for the first time. This efficient protoplast isolation and transformation protocol is sufficient for studies on protein subcellular localization and protein–protein interaction, and shall be suitable for many other molecular biology applications where the mesophyll protoplast system is desirable in perennial ryegrass.

Establishment of transient gene expression systems in protoplasts from Liriodendron hybrid mesophyll cells.

Abstract: Liriodendron is a genus of the magnolia family comprised of two flowering tree species that produce hardwoods of great ecological and economic value. However, only a limited amount of genetic research has been performed on the Liriodendron genus partly because transient or stable transgenic trees have been difficult to produce. In general, transient expression systems are indispensable for rapid, high-throughput screening and systematic characterization of gene functions at a low cost; therefore, development of such a system for Liriodendron would provide a necessary step forward for research on Magnoliaceae and other woody trees. Herein, we describe an efficient and rapid protocol for preparing protoplasts from the leaf mesophyll tissue of a Liriodendron hybrid and an optimized system for polyethylene glycol-mediated transient transfection of the protoplasts. Because the leaves of the Liriodendron hybrid are waxy, we formulated an enzyme mix containing 1.5% (w/v) Cellulase R-10, 0.5% (w/v) Macerozyme R-10, and 0.1% (w/v) Pectolyase Y-23 to efficiently isolate protoplasts from the Liriodendron hybrid leaf mesophyll tissue in 3 h. We optimized Liriodendron protoplast transfection efficiency by including 20 μg plasmid DNA per 104 protoplasts, a transformation time of 20 min, and inclusion of 20% (w/v) polyethylene glycol 4000. After integrating the Liriodendron WOX1 gene into pJIT166-GFP to produce a WOX1-GFP fusion product and transfecting it into isolated protoplasts, LhWOX1-GFP was found to localize to the nucleus according to its green fluorescence.

Construction of Potent Recombinant Strain Through Intergeneric Protoplast Fusion in Endophytic Fungi for Anticancerous Enzymes Production Using Rice Straw

Abstract: Among all fungal endophytes isolates derived from different ethno-medical plants, the hyper-yield L-asparaginase and L-glutaminase wild strains Trichoderma sp. Gen 9 and Cladosporium sp. Gen 20 using rice straw under solid-state fermentation (SSF) were selected. The selected strains were used as parents for the intergeneric protoplast fusion program to construct recombinant strain for prompt improvement production of these enzymes in one recombinant strain. Among 21 fusants obtained, the recombinant strain AYA 20-1, with 2.11-fold and 2.58-fold increase in L-asparaginase and L-glutaminase activities more than the parental isolates Trichoderma sp. Gen 9 and Cladosporium sp. Gen 20, respectively, was achieved using rice straw under SSF. Both therapeutic enzymes L-asparaginase and L-glutaminase were purified and characterized from the culture supernatant of the recombinant AYA 20-1 strain with molecular weights of 50.6 and 83.2 kDa, respectively. Both enzymes were not metalloenzymes. Whereas thiol group blocking reagents such as p-chloromercurybenzoate and iodoacetamide totally inhibited L-asparaginase activity, which refer to sulfhydryl groups and cysteine residues involved in its catalytic activity, they have no effect toward L-glutaminase activity. Interestingly, potent anticancer, antioxidant, and antimicrobial activities were detected for both enzymes.

Detection of Oil Palm Root Penetration by Agrobacterium-Mediated Transformed Ganoderma boninense, Expressing Green Fluorescent Protein

Abstract: A highly efficient and reproducible Agrobacterium-mediated transformation protocol for Ganoderma boninense was developed to facilitate observation of the early stage infection of basal stem rot (BSR). The method was proven amenable to different explants (basidiospore, protoplast, and mycelium) of G. boninense. The transformation efficiency was highest (62%) under a treatment combination of protoplast explant and Agrobacterium strain LBA4404, with successful expression of an hyg marker gene and gus-gfp fusion gene under the control of heterologous p416 glyceraldehyde 3-phosphate dehydrogenase promoter. Optimal transformation conditions included a 1:100 Agrobacterium/explant ratio, induction of Agrobacterium virulence genes in the presence of 250 μm acetosyringone, co-cultivation at 22°C for 2 days on nitrocellulose membrane overlaid on an induction medium, and regeneration of transformants on potato glucose agar prepared with 0.6 M sucrose and 20 mM phosphate buffer. Evaluated transformants were able to infect root tissues of oil palm plantlets with needle-like microhyphae during the penetration event. The availability of this model pathogen system for BSR may lead to a better understanding of the pathogenicity factors associated with G. boninense penetration into oil palm roots.

Protoplast Swelling and Hypocotyl Growth Depend on Different Auxin Signaling Pathways.

Abstract: Members of the TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX PROTEIN (TIR1/AFB) family are known auxin receptors. To analyze the possible receptor function of AUXIN BINDING PROTEIN1 (ABP1), an auxin receptor currently under debate, we performed different approaches. We performed a pharmacological approach using α-(2,4-dimethylphenylethyl-2-oxo)-indole-3-acetic acid (auxinole), α-(phenylethyl-2-oxo)-indole-3-acetic acid (PEO-IAA), and 5-fluoroindole-3-acetic acid (5-F-IAA) to discriminate between ABP1- and TIR1/AFB-mediated processes in Arabidopsis (Arabidopsis thaliana). We used a peptide of the carboxyl-terminal region of AtABP1 as a tool. We performed mutant analysis with the null alleles of ABP1, abp1-c1 and abp1-TD1, and the TILLING mutant abp1-5. We employed Coimbra, an accession that exhibits an amino acid exchange in the auxin-binding domain of ABP1. We measured either volume changes of single hypocotyl protoplasts or hypocotyl growth, both at high temporal resolution. 5-F-IAA selectively activated the TIR1/AFB pathway but did not induce protoplast swelling; instead, it showed auxin activity in the hypocotyl growth test. In contrast, PEO-IAA induced an auxin-like swelling response but no hypocotyl growth. The carboxyl-terminal peptide of AtABP1 induced an auxin-like swelling response. In the ABP1-related mutants and Coimbra, no auxin-induced protoplast swelling occurred. ABP1 seems to be involved in mediating rapid auxin-induced protoplast swelling, but it is not involved in the control of rapid auxin-induced growth.

Protoplast fusion in the genus Gentiana : genomic composition and genetic stability of somatic hybrids between Gentiana kurroo Royle and G. cruciata L.

Abstract: Somatic hybridization by protoplast fusion is used in breeding programs to obtain plant material that has inherited valuable traits from two different species, and in order to broaden plant genetic diversity. Somatic hybrids of the genus Gentiana could provide a useful source of new ornamental cultivars and of secondary metabolites. However, in order to evaluate its further usefulness, detailed characterization of the newly created hybrid is essential. Here, genome composition and stability of interspecific gentian somatic hybrids obtained following electrofusion of cell suspension-derived protoplasts of diploid Gentiana kurroo Royle with leaf mesophyll-derived protoplasts of tetraploid G. cruciata L. were characterized using various molecular markers and flow cytometry. AFLP and ISSR analyses revealed that all 21 hybrid plants and 3 lines of hybrid callus were genetically closer to G. cruciata than to G. kurroo. According to the results of chloroplast DNA analysis with the use of CAPS markers, all somatic hybrids inherited chloroplasts from “mesophyll” fusion partner G. cruciata. Flow cytometry revealed that polyploidization occurred, and probably it took place at the early stage of post-fusion culture. In consequence, gradual elimination of nuclear DNA, mixoploidy, and high genetic instability were observed in most hybrid plants and calli during the subsequent 4 years of in vitro culture.

Plasmolysis-deplasmolysis causes changes in endoplasmic reticulum form, movement, flow, and cytoskeletal association.

Abstract: Plasmolysis of hypocotyl cells of transgenic Arabidopsis thaliana and Nicotiana benthamiana diminishes the dynamics of the remodeling of the endoplasmic reticulum (ER) in the central protoplast, namely that withdrawn from the cell wall, and more persistent cisternae are formed, yet little change in the actin network in the protoplast occurs. Also, protein flow within the ER network in the protoplast, as detected with fluorescence recovery after photobleaching (FRAP), is not affected by plasmolysis. After plasmolysis, another network of strictly tubular ER remains attached to the plasma membrane-wall interface and is contained within the Hechtian strands and reticulum. FRAP studies indicate that protein flow within these ER tubules diminishes. Actin is largely absent from the Hechtian reticulum and the ER becomes primarily associated with altered, branched microtubules. The smaller volume of the central protoplast is accompanied by decreased movement rates of tubules, cisternae, and spheroid organelles, but this reduced movement is not readily reversed by the increase in volume that accompanies deplasmolysis.

In Vitro Bioassay of Allelopathy in Four Bamboo Species; Bambusa multiplex, Phyllostachys bambusoides, P. nigra, Sasa kurilensis, Using Sandwich Method and Protoplast Co-Culture Method with Digital Image Analysis

Abstract: Moderately strong allelopathic activities were found in four bamboo species, Bambusa multiplex cv. Houraichiku; Phyllostachys bambusoides cv. Madake; P. nigra cv. Hachiku; Sasa kurilensis cv. Chishimazasa, which are of different classification or of different ecological distributions, using the “Sandwich Method”, which assays the dried leaves on growth of lettuce seedlings. Only small difference of activity was found among the four bamboo species. In addition, “Protoplast Co-culture Method” for assay of allelopathy in a 50 μL liquid medium using a 96 well culture plate, was applied to the suspension cultures of the four bamboo species. Protoplasts were isolated from two-week cultured suspension cells of four bamboo species using Cellulase RS and Pectolyase Y-23 in 0.6 M mannitol. At low protoplast densities of bamboo, B. multiplex and P. bambusoides stimulated the recipient lettuce growth, i.e., non-spherically cell enlargement and cell divisions observed under an inverted microscope, while protoplasts of P. nigra and S. kurilensis were less stimulatory or inhibitory. Inhibitory effect of S. kurilensis was the strongest among four bamboo species. Furthermore, highly inhibitory effects of S. kurilensis protoplasts on yellow color accumulation of lettuce protoplasts were clearly observed by analysis of a scanned digital image of a 96-well culture plate. Differences and causes of the allelopathic activities were discussed comparing with other plant species studied using the same assay methods.

Enhanced production of fructosyltransferase in Aspergillus oryzae by genome shuffling.

Abstract: To breed Aspergillus oryzae strains with high fructosyltransferase (FTase) activity using intraspecific protoplast fusion via genome-shuffling. A candidate library was developed using UV/LiCl of the conidia of A. oryzae SBB201. By screening for enzyme activity and cell biomass, two mutants (UV-11 and UV-76) were chosen for protoplast fusion and subsequent genome shuffling. After three rounds of genome recombination, a fusion mutant RIII-7 was obtained. Its FTase activity was 180 U g−1, approximately double that of the original strain, and RIII-7 was genetically stable. In fermentation culture, FTase activity of the genome-shuffled strain reached a maximum of 353 U g−1 using substrate-feeding method, and this value was approximately 3.4-times higher than that of the original strain A. oryzae SBB201. Intraspecific protoplast fusion of A. oryzae significantly enhanced FTase activity and generated a potentially useful strain for industrial production.

Old methods rediscovered: application and improvement of two direct transformation methods to hybrid poplar (Populus tremula × P. alba)

Abstract: Two direct DNA transfer methods, biolistic transformation and a protoplast transformation approach using the INRA-clone 717 1B4 (Populus tremula × P. alba), are applied to poplars and compared. Both the in vitro culture and the transformation parameters were optimized to receive a maximum quantity of transformed cells to achieve a stable transformation. For the first time, the stable integration of gfp and dsred in the poplar genome and their expression as visual reporter genes in regenerated plantlets can be shown. For biolistic transformation, stem segments cut lengthwise and incubated for 10 days on a callus induction medium revealed the highest number of transient Gfp- and dsRed signals. After optimization of the in vitro culture parameter, Gfp and dsRed-expressing transgenic poplars were regenerated, proven by PCR and Southern blot analysis. For protoplast transformation, the focus was initially on the development of a highly efficient protoplast isolation and plant regeneration system. Using an enzyme solution consisting of 1.0% cellulase R10 and 0.24% macerozyme, 1 × 107 protoplasts were obtained from 1 g fresh weight leaves. Following incubation of the protoplasts in 600 mOsm culture medium, a high number of microcalli were obtained, from which plantlets were regenerated. The parameters for isolation and regeneration were then complemented by an efficient protoplast transformation protocol with 40% PEG1500. The results of this study confirm that both the biolistic and the protoplast transformation methods can be considered suitable for transferring cisgenes directly into poplar.

In vitro bioassay of allelopathy of Arabidopsis thaliana by sandwich method and protoplast co-culture method with digital image analysis

Abstract: We examined the allelopathic activities of Arabidopsis thaliana, ecotype Columbia by two in vitro methods. The effect of dried leaves on the growth of recipient lettuce seedlings was examined by the sandwich method. The allelopathic activity on protoplast growth was examined by co-culture with recipient lettuce leaf protoplasts in 50 µl liquid medium using a 96-well culture plate. Non-spherically enlarged or dividing protoplasts of lettuce were counted under an inverted microscope. Inhibition of yellow accumulation during lettuce protoplast growth was quantitated by image analysis of scanned digital images of 96-well culture plates. The results were described as the percentages of control without A. thaliana. The results were compared with those obtained using several plants which had strong allelopathic activities on recipient lettuce using the same methods. The growth of lettuce protoplasts was inhibited at both 4 and 8 days of culture with protoplasts of A. thaliana. The results suggested the usefulness of the protoplast co-culture method for future studies on allelopathy.

Dendrobium protoplast co-culture promotes phytochemical assemblage in vitro

Abstract: The present study is intended to analyze the occurrence of potent, low produce, naturally occurring stilbenes in protoplasts of wild species and hybrids of Dendrobium. The wild species selected for the study was Dendrobium ovatum, endemic to Western Ghats of India. Protoplasts were isolated from leaves and tepal tissues of all the species and were cultured purely to generate homofusants and cross-cultured to raise heterofusants. Phytochemical composition of protoplast culture with atypical and pure microcolonies was performed using mass spectrometry. Enzyme cocktail of 4% pectinase together with 2% cellulase displayed the highest competence for protoplast isolations. Maximum protoplast density of 30.11 × 104/ml was obtained from D. ovatum leaves in 2 h. Subcellular features such as the presence of partially formed cell wall, the position of the nucleus, chloroplast density, colony existence, and integrity of the plasma membrane were analyzed. Among the pure and cross-cultured protoplasts, the number of heterofusants and homofusants formed were enumerated. The spectral feature extraction of the mass spectrometry indicated the presence of five phenolic marker compounds, viz., tristin, confusarin, gigantol, moscatilin, and resveratrol, some of them in pure and others in assorted protoplast cultures raised from Dendrobium leaves and tepals. The study demonstrated that protoplast fusion technique enabled phytochemical assemblage in vitro as stilbenes tend to get restricted either in a tissue or species specific manner. This is the first report showing the presence of resveratrol, moscatilin, tristin, gigantol, and confusarin in wild and hybrid species from cultured Dendrobium protoplasts in vitro.

Simple and reliable system for transient gene expression for the characteristic signal sequences and the estimation of the localization of target protein in plant cell.

Abstract: The efficiency of transient gene expression in plants credibly demonstrated characteristics of gene functions in numerous studies. Two key strategies of transient expression became favorites among researchers: protoplast transfection and agroinfiltration. Each of them, alongside the advantages, has its own constraints. In this work, an easy, rapid, and reliable system for characterization of the signal sequences and determinations of target protein localization in a plant cell is proposed and tested. This system—called the AgI–PrI—implies production of protoplasts from plant tissues after agroinfiltration. Reliability of the proposed system for transient gene expression has been proved using characterized signal sequences in Nicotiana benthamiana cells. The corresponding protocol is less expensive and depends to a lesser degree on the professional skills in the area of protoplast isolation and transfection; furthermore, it may be applicable to other plant species with either available efficient methods of agroinfiltration and protoplast isolation or with the potential for one of the protocols to be supplemented. Thus, the AgI–PrI technique makes it possible to combine the advantages of two widely used methods for the transient gene expression in plants—agroinfiltration and protoplast isolation and transfection—and concurrently avoids their critical points.

Early studies on the effect of peptide growth factor phytosulfokine-α on Brassica oleracea var. capitata L. protoplasts

Abstract: Phytosulfokines (PSK) are peptidyl growth factors with the potential of inducing cell proliferation. We examined the effect of supplementation of liquid culture medium with 0.1 µM phytosulfokine-α (PSK-α) on protoplast viability and division frequencies in seven accessions of Brassica oleracea var. capitata L., including cultivars and breeding lines. Protoplasts were isolated from leaves and hypocotyls of in vitro grown plants and immobilized in calcium-alginate layers. Cabbage protoplast-derived cells cultured in medium supplemented with 0.1 µM of PSK-α had higher viability and division frequencies compared to cells cultured in PSK-α-free control medium. The effect of PSK-α was more pronounced in low-responding accessions (‘Slawa z Golebiewa’, ‘Ramkila F1’, LM, and LM98); however, in two cultivars with very low response (‘Badger Shipper’ and ‘Oregon 123’), although the division frequencies in the media supplemented with PSK-α were increased over the control, the differences were not significant. Obtained callus colonies were subjected to regeneration. PSK-α supplemented into the liquid culture medium had an indirect effect on shoot regeneration by inducing sustained cell divisions leading to an increase in shoot regeneration in Slawa z Golebiewa and both breeding lines.

Regeneration of protoplasts after somatic hybridisation of Hydrangea

Abstract: Somatic hybridisation of Hydrangea is a promising tool to obtain new basic material for breeding. Viable mesophyll protoplasts were isolated from 21 cultivars and accessions of H. macrophylla, H. paniculata, H. arborescens, H. quercifolia and H. febrifuga. Induction of cell divisions was observed after electromanipulation and fusion by polyethylene glycol. Multi-cellular structures developed in liquid media. Plated microcalli grew on different solid regeneration media. Several phytohormones, their concentration and combination influenced the development of callus and roots. The regular appearance of endophytes challenged successful plant regeneration. As a result of endophytes, the development of microcolonies stopped and they died in liquid protoplast media. Plated microcalli or growing calli turned brown on regeneration medium. Antibiotic Timentin® inhibited expansion of endophytes for a short time. The addition of ascorbic and citric acid to the regeneration media had inhibiting effect on endophyte growth. Calli showed more vitality and grew faster. The supplement of the regeneration media with karrikinolide, a recently discovered new plant growth regulator, brought contradictory results. After addition of karrikinolide microcolonies looked healthier by their shining green colour in liquid media followed by a severe browning of callus soon afterwards. In the further course, karrikinolide promoted the development of endophytes. Shoot induction and plant regeneration succeeded only once from callus that was a result from H. macrophylla ‘Schneeball’ and H. macrophylla ‘Nachtigall’ fusion.

Functional characterization of plasma membrane-localized organic acid transporter (CsALMT1) involved in aluminum tolerance in Camelina sativa L.

Abstract: Aluminum (Al) toxicity is a major limiting factor that inhibits root elongation and decreases crop production in acidic soils. The symptoms of inhibited root growth include a reduced uptake of nutrients because the roots become stubby and brittle. The release of organic anions from roots can protect a plant from Al toxicity. The mechanism relies on the efflux of organic anions, such as malate or citrate, which protect roots by chelating the Al3+. In this study, homologs of TaALMT1, a Camelina gene that encodes an aluminum-activated malate transporter, were investigated. The expression of this gene was induced by Al in the root, but not in the shoots. Using green fluorescent protein (GFP) fusion constructs and Western-blot analysis, we observed that CsALMT1 was localized in the plasma membrane. Also, to determine the degree to which Al tolerance was affected by malate secretion in Camelina root, we generated CsALMT1 overexpressing plants. CsALMT1 overexpressing transgenic plants showed a higher root elongation rate than the wild-type plant. Damaged cell staining analysis by hematoxylin under 25 µM Al treatment for 2, 4, and 6 h showed a pattern of less damage in CsALMT1 transgenic plants than in wild-type plant, especially in the root elongation zone. Furthermore, the rate of increase of secretion of organic acid in overexpressed plants after Al treatment was higher than that in the wild-type plant. In addition, in the Al-specific dye morin staining on root protoplast under 50 µM Al treatment, less Al accumulation was observed in the CsALMT1 transgenic plants than in the wild-type plant. The Al contents in the roots of the transgenic plants were at a lower level than those in the wild-type plant. These results show that the overexpression of CsALMT1 improves Al tolerance by increasing the release of malate from the root to the soil and, thereby, detoxifies the Al3+.

Regeneration of cell suspension derived Apium graveolens L. protoplasts

Abstract: Cytoplasmatic male sterility (CMS), which can be achieved by protoplast fusion and regeneration, has potential to greatly facilitate hybrid breeding of celery (Apium graveolens L.). Therefore as a first step we developed a simple and efficient protoplast isolation and regeneration protocol for three commercial A. graveolens varieties (green and white celery and celeriac). To this end, cell suspensions from independent cell lines of open pollinated cultivars and inbred lines were initiated as a source for protoplast isolation. Comparative analyses showed that culturing was most successful in modified Kao and Michayluk liquid medium supplemented with 0.3 mg l−1 2,4-D. The cytokinin type (TDZ or zeatin) and concentration had no significant effect on regeneration efficiency. Microcalli were obtained within 15 days to 5 weeks after protoplast isolation. Supplementing the culture medium with 25% conditioned medium increased microcolony formation for some of the cultured lines. Plants were obtained within 2 months of microcallus culturing and these were all diploid, suggesting genetic inheritance consistency. The efficiency of regeneration mainly depended on the specific genotype, with outcrossing genotypes displaying high heterogeneity in regeneration responses whereas inbred lines did not regenerate. The protocol presented here enables to implement protoplast fusion in celery breeding.

Improvement of nutrition production by protoplast fusion techniques in Chlorella vulgaris

Abstract: Recent decades showing remarkable development of the biotechnology of microalgae.Valuable product for food, nutrition and other applications will extend into broader area. Natural nutrition production from microalgae are not yet competitive with their synthetic levels. Chlorella is widely used as a health food and feed supplement, as well as in the pharmaceutical and cosmetics industries. Protoplast fusion was found to be an efficient method in improving its nutrition production and diversification in Chlorella vulgaris. The research was carried out by application of protoplast fusion on interspecific microalgae of C. vulgaris. The fusant was subjected for analysis of nutrition content by GCMS methods on C. vulgaris powder from 100 L liquid cultivation of fusant. The research result gained fusant in high mass production level. Nutrition analysis of fusants showed 17 amino acid with high concentration glutamic acid (14495.52 ppm) followed by leucine (10856.97 ppm) and Aspartic acid (10378 ppm). Palmitic acid (1.59%) was showed highest concentration in its lipid acid profile. Lipid analysis also showed polyunsaturated fatty acids (PUFA) with concentration 1.0987% and DHA 0.2%. Surprisingly, the fusant also revealed Omega 9 instead of Omega 3 and Omega 6. The research result showed potential acquisition of improvement nutrition by protoplast fusian application on microalgae.

Method and special culture medium for efficiently separating, converting and regenerating potato protoplast

Abstract: The invention discloses a method and special culture medium for efficiently separating, converting and regenerating a potato protoplast. The method comprises the steps of carrying out induction and three-time subculture on a starting material, namely a stem segment of a potato test-tube seedling to obtain an embryogenic callus vigorous in growth and loosened in structure, and carrying out suspension culture; slaking potato suspension cells by using cellulases, pectases and macerases; and separating a protoplast by using a density gradient sedimentation method to obtain a high-purity protoplast. A target gene is introduced to a potato protoplast genome through polyethylene glycol mediation, and liquid shallow culture and subsequent differentiation regeneration are performed, so that a great number of transgenic plants of potatoes are cultured.

Establishment of Callus Induction and Cell Suspension Cultures of Dendrathema Indicum var. Aromaticum a Scented Chrysanthemum

Abstract: Dendranthema indicum var. aromaticum is an important aroma plant in genus Dendrathema, and the establishment of callus cultures and cell suspension cultures is the basement of further protoplast fusion studies, which make it possible to breed new fragrant chrysanthemum. In this study, the effects of different plant growth regulating substances in different concentrations on callus induction were investigated with stem segments, leaves, petioles as explants. The results showed that the optimal explants were lower stem segments according to the percentage of callus formation, callus hardness, growth potential and shoot differentiation. The optimal induction mediums were MS supplemented with 1.0 mg．l-12.4 D and 0.2 mg．l-1 6-BA. The cell suspension culture system was established by using the subculture calli. The results showed that the suitable inoculum size was 2g and the suitable cell suspension culture medium was MS supplemented with 0.2 mg．l-1 6-BA and 0.5 mg．l-1 2,4-D. The time course of cell growth showed that the greatest cell fresh weight appeared on day 14 and the highest cell viability on day 3.

Nitraria sibirica cell suspension culture: establishment, characterization and application

Abstract: Nitraria sibirica Pall. is a shrub that grows in saline-alkali soil and has traditional medicinal value and potential commercial value. The objectives of this study include induction and multiplication of callus, establishment of a suspension cell line, and isolation of protoplasts from cell suspensions. Murashige and Skoog (MS) medium was used for callus induction from mature seeds of N. sibirica. Seed-derived calluses were further multiplied on MS medium augmented with 0.5 mg L−1 6-benzylaminopurine (6-BA) and 1.0 mg L−1 2,4-dichlorophenoxy (2,4-D) acetic acid. Suspension cultures of N. sibirica were initiated by transferring friable calli to the same liquid multiplication medium. Characterization of the suspension culture was assessed based on fresh mass, dry mass, cell viability and pH value of the culture. A typical growth curve was observed after inoculating 1.5 g of callus in 40 mL liquid medium, including a lag phase, an exponential growth phase, a stationary phase, and a negative acceleration phase. The effect of factors such as pre-plasmolysis, enzyme combination, enzymolysis time and mannitol concentration, on the isolation of cell-derived protoplasts were evaluated to determine the usefulness of suspension cultures. The maximum yield (9.79 × 106 cells/g) and highest viability (79.97%) of protoplast were reached when approximately 1 g of cell suspension (cultured for 6 days) was inoculated for 12 h in cell and protoplast washing solution made of 0.8 mol L−1 mannitol mixture solution, cellulose onozuka R-10 2% (w/v), hemicellulose 0.2%, macerozyme R-10 1%, and pectolyase Y-23 0.5%. Protoplast yield was significantly influenced by pre-plasmolysis and cellulose onozuka R-10 (P < 0.05).

Establishment of morphogenic rice cell suspension cultures (Oryza sativa) in Costa Rica

Abstract: The optimization of lhe in vitro culture of cereals forms lhe basis for the genetic transformation of rice. The establishment of embryogenic cell suspension cultures is essential for the development of more advanced methods of genetic engineering, such a genetic Lransfonnalion of plant cells and somatic hybridization through protoplast fusion.

Method for preparing and purifying cedarwood protoplast

Abstract: The invention discloses a method for preparing and purifying cedarwood protoplast The method comprises the following steps of: selecting laminae or tender stems on the uppermost part of a cedarwood tissue culture seedling, which are not subjected to separating seedling; cutting the laminae or stems, adding into enzymatic hydrolysate, vacuumizing, and putting on a horizontal shaking table to carry out enzymolysis at the dark place for 2- 4 h; filtering enzymatic hydrolysate by using a cell strainer with a bore diameter of 70Mum to a centrifugal tube, cleaning the laminae or stems which are subjected to enzymolysis by using protoplast cleanout fluid, filtering by using the cell strainer with the bore diameter of 70Mum, merging filtrate into the same centrifugal tube, and centrifuging to remove supernatant; adding the pre-cooled protoplast cleanout fluid into a precipitate to wash protoplast, centrifuging to remove supernatant, and repeatedly washing, resuspending the collected precipitate by using protoplast resuspending solution, and obtaining the purified cedarwood protoplast By the method, the yield of the protoplast of the cedarwood laminae is 1 8x106/g, and the vitality is 94%; and the yield of the protoplast of the cedarwood stems is 8 9x105/g and the vitality is 86 5% The acquired protoplast is high in yield and vitality